The misfolding of different cellular proteins into amyloid-like aggregates is associated with non- infectious neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's, as well as with the infectious prion diseases e.g. Mad Cow, Chronic Wasting in deer and Creutzfeldt-Jacob in humans. For each of these diseases, the associated protein aggregate ("seed') attracts its normal conformers to misfold and join the aggregate. Certain proteins in the simple eukaryote yeast, can likewise misfold into infectious amyloid aggregates, and these aggregates cause epigenetic variation. In this proposal, the power of yeast genetics and molecular biology is used to study how proteins misfold into amyloid-like aggregates and the consequences of this misfolding for the cell. The extensive similarity between yeast and human cells, which has enabled yeast models to make significant contributions in understanding human disease, implies that these studies will likely be relevant to misfolded aggregating proteins in humans. Since most human protein misfolding diseases occur without infection by any external seed, Aim I focuses on the molecular mechanisms surrounding spontaneous cellular amyloid formation. The questions addressed are: where do newly appearing prion aggregates first arise in cells, what other proteins are associated with them, and how do pre-existing prions enhance the de novo appearance of heterologous prions? Aim I also tests the hypothesis that Sla2, the yeast homolog of the mammalian huntingtin interacting protein, facilitates the ability of existing prions to cross-seed the de novo aggregation of heterologous prion proteins, by binding to both the seed and protein to be seeded, thereby placing them in close proximity. Interestingly, human and yeast prion proteins can each form multiple variants of amyloid aggregates that differ in structure and cause distinct phenotypes or disease pathologies, even though the amino acid sequences of the proteins are identical. Aim II identifies proteins bound to, and/or required for, the propagation of several prions and their variants. In addition, solid-state NMR structures of two variants of the same prion will be determined with the help of collaborators. By comparing heterologous prions, as well as different variants of the same prion, factors likely to be common to the maintenance and infectivity of all prions and that should therefore provide useful drug targets, will be identified. While amyloid formation is associated with disease, the actual cause of pathology is unclear. In Aim III, genetic and molecular studies of two prions that cause toxicity in yeast will help define the toxic species. Finally, yeast prions are important not only as a model for human disease, but also because they suggest an important new mechanism of genetic variation operating at the level of protein conformation rather than nucleic acids. In Aim IV the fascinating question of whether prions can sometimes provide the host cell with an advantage is explored, along with the possibility that such advantageous prions may also exist in mammals. PUBLIC HEALTH RELEVANCE: The power of yeast genetics and molecular biology will be used to study the misfolding of proteins into amyloid-like aggregates like those associated with several devastating neurodegenerative human diseases including Alzheimer's, Parkinson's, Huntington's and Creutzfeldt-Jacob's diseases. The extensive similarity between yeast and human cells, which has enabled yeast models to make significant contributions in understanding other human diseases, implies that the insights gained from these studies will be helpful when choosing drug-targets for the human diseases.